The invention relates to novel nucleic acid sequences which encode human autoantibodies against blood platelet membrane proteins and which encode antiidiotypic antibodies, to novel amino acid sequences of human antibodies, and to their use for the diagnosis and therapy of diseases.
Autoimmune thrombocytopenic purpura (AITP) is an immune disease which is defined by a low blood platelet count associated with normal or elevated megakaryocyto-poiesis. The destruction of platelets in the reticuloendothelial system (spleen, liver and bone marrow) is increased due to the presence of anti-platelet autoantibodies. These autoantibodies, which can be detected in about 75% of AITP patients, are predominantly directed against the platelet membrane glycoproteins (GP) IIb/IIIa and Ib/IX. Several different autoantibody specificities may be found in one and the same patient (cf., e.g., Berchtold and Wenger, Blood 81 (1993), 1246-1250; Kiefel et al., Br. J. Haematol. 79 (1991), 256-262; McMillan et al., Blood 70 (1987), 1040 and Fujisawa et al., Blood 79 (1991); 1441). However, it is still difficult to characterize binding epitopes and to ascertain the pathogenic significance of the autoantibodies due to the limited quantity of autoantibodies which can be obtained from AITP patients. It has only been possible to obtain a few human monoclonal antibodies from lymphocytes of AITP patients which react with GPIIb/IIIa AIPT using the hybridoma technique (Kunicki et al., Hum. Antibodies Hybridiomas 1(1990) 83-95).
Natural autoantibodies against various selfantigens, for example against intracellular and cytoskeletal components of human platelets, have also been reported to occur in healthy individuals (Guilbert et al., J. Immunol. 128 (1982), 2779-2787; Hurez et al., Eur. J. Immunol. 23 (1993), 783-789 and Pfueller et al., Clin. Exp. Immunol. 79 (1990), 367-373). Some of these autoantibodies which have been observed in sera from healthy individuals can also be directed against platelet-membrane proteins (Souberbielle, Eur. J. Haematol. 56 (1996), 178-180). However, the role of these natural autoantibodies, and there relationship to disease-associated autoantibodies, is still unknown.
Corticosteroids can be used for treating AITP. About half of the patients react within 4 weeks to an administration of prednisone; however long-term remissions are only rarely seen. The administration of high doses of intravenous immunoglobulin (IVIgG) is recommended as an emergency treatment for patients who are exhibiting severe bleeding or extremely low platelet counts. This treatment is followed in most patients by a rapid, but usually only transient, increase in the platelet count. The mechanisms by which corticosteroids and IVIgG act in the treatment of AITP are still unknown. Investigations carried out by Berchtold et al., (Blood 74 (1989), 2414-2417 and Berchtold and Wenger, Blood 81 (1993), 1246-1250) have disclosed that antiiodiotypic antibodies which are present in IVIgG can inhibit the binding of autoantibodies to platelet glycoproteins.
The problem underlying the present application is that of identifying novel DNA sequences which are responsible for autoantibodies binding to GPIIb/IIIa. This approach can be used for making available novel pharmaceutical preparations which can be employed for improving the diagnosis and therapy of AITP.
It was surprisingly possible to identify binding sequences from autoantibodies after using peripheral circulating B cells from a healthy human donor to prepare a combinatorial phagemid display library of human antibody heavy and light chains. Following the presentation of human heavy and light antibody Fab fragments on the surface of the filamentous phage M13, it was possible to identify phage clones which exhibit specific binding to GPIIb/IIIa.
For this, the phagemid library was brought consecutively into contact with thrombasthenic platelets lacking GPIIb/IIIa (negative selection) and normal platelets (positive selection). After several rounds of selection and amplification by infecting E.coli, 23 clones were obtained which were able to bind to the GPIIb/IIIa complex. Inhibition studies using pools of monoclonal antibodies directed against the GPIIb/IIIa yielded two groups of clones: both groups were inhibited by monoclonal antibodies which were specific for the GPIIb/IIIa complex and one group was also inhibited by a GPIIb-specific monoclonal antibody. These findings were confirmed by carrying out a DNA analysis of the clones which indicated the presence of 2 different anti-GPIIb/IIIa phage clones. These results demonstrate that 2 GPIIb/IIIa-specific phage clones, i.e. autoantibodies, can be cloned from the genome of a healthy individual and that these clones are able to recognize confirmational epitopes belonging to the GPIIb/IIIa complex. Inhibition studies furthermore established that both phage clones inhibit the binding of platelet-associated autoantibodies from AITP patients to purified GPIIb/IIIa and therefore presumably recognize GPIIb/IIIa epitopes which are AITP-associated. Since the phage clones contain the antigen-binding sequences of natural autoantibodies which are derived from the genome of a healthy individual, this finding can lead to new insights into the origin of platelet-associated autoantibodies in AITP.
In addition to this, it is possible to use the novel phage clones to produce recombinant antiidiotypic antibodies against anti-GPIIb/IIIa autoantibodies, with the anti-GPIIb/IIIa phage clones being used as antigen. The recombinant antiidiotypic antibodies which can be obtained in this way constitute an attractive clinical alternative to using IVIgG.
The nucleotide sequences of the identified phage clones, and the amino acid sequences which are deduced from these nucleotide sequences, are depicted in the sequencing listings SEQ ID No. 1 to 8 (autoantibodies) and SEQ ID No. 9 to 18 (antiidiotypic antibodies).
I. Autoantibodies
A first aspect of the present invention relates to nucleic acids which encode auto-antibodies. Part of the subject-matter of the invention is therefore a nucleic acid which encodes the heavy chain of a human antibody, or a functional derivative or a fragment thereof, and encompasses a CDR3 region, selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
VLPFDPISMDV, (SEQ ID NO:31)
(b) a nucleotide sequence which encodes the amino acid sequence:
ALGSWGGWDHYMDV, (SEQ ID NO:32)
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a) or (b), and
(d) a nucleotide sequence which encodes an amino acid sequence having an equivalent ability to bind to GPIIb/IIIa.
The novel nucleic acid furthermore preferably comprises a CDR1 region selected from:
(a) a nucleotide sequence which encodes the amino acid sequence: GYSWR, (SEQ ID NO:33)
(b) a nucleotide sequence which encodes the amino acid sequence: SYAMH, (SEQ ID NO:34)
and
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a) or (b).
The novel nucleic acid preferably furthermore comprises a CDR2 region selected from:
(a) a nucleotide sequence which encodes th amino acid sequence:
DISYSGSTKYKPSLRS, (SEQ ID NO:35)
(b) a nucleotide sequence which encodes the amino acid sequence:
VISYDGSNKYYADSVKG, (SEQ ID NO:36)
and
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably of at least 90%, with an amino acid sequence from (a) or (b).
A second aspect of the present invention is a nucleic acid which encodes the light chain of a human antibody, or a functional derivative or a fragment thereof, and comprises a CDR3 region, selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
ATWDDGLNGPV, (SEQ ID NO:37)
(b) a nucleotide sequence which encodes the amino acid sequence:
AAWDDSLNGWV, (SEQ ID NO:38)
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably of at least 90%, with an amino acid sequence from (a) or (b), and
(d) a nucleotide sequence which encodes an amino acid sequence having an equivalent ability to bind to GPIIb/IIIa.
The novel nucleic acid preferably furthermore comprises a CDR1 region selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
SGSSSNIRSNPVS, (SEQ ID NO:39)
(b) a nucleotide sequence which encodes the amino acid sequence:
SGSSSNIGSNTVN, (SEQ ID NO:40)
and
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a) or (b).
In addition, the novel nucleic acid preferably further comprises a CDR2 region selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
GSHQRPS, (SEQ ID NO:41)
(b) a nucleotide sequence which encodes the amino acid sequence:
SNNQRPS, (SEQ ID NO:42)
and
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a) or (b).
II. Antiidiotypic antibodies
A second aspect of the present invention relates to nucleic acids which encode antiidiotypic antibodies. Part of the subject-matter of the invention is therefore a nucleic acid which encodes the heavy chain of a human antibody, or a functional derivative or a fragment thereof, and comprises a CDR3 region, selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
VRDLGYRVLSTFTFDI, (SEQ ID NO:43)
(b) a nucleotide sequence which encodes the amino acid sequence:
DGRSGSYARFDGMDV, (SEQ ID NO:44)
(c) a nucleotide sequence which encodes the amino acid sequence:
MGSSVVATYNAFDI, (SEQ ID NO:45)
(d) a nucleotide sequence which encodes the amino acid sequence:
DADGDGFSPYYFPY, (SEQ ID NO:46)
(e) a nucleotide sequence which encodes the amino acid sequence:
LRNDGWNDGFDI, (SEQ ID NO:47)
(f) a nucleotide sequence which encodes the amino acid sequence:
DSETAIAAAGRFDI, (SEQ ID NO:48)
(g) a nucleotide sequence which encodes the amino acid sequence:
EDGTTVPSQPLEF, (SEQ ID NO:49)
(h) a nucleotide sequence which encodes the amino acid sequence:
GSGSYLGYYFDY, (SEQ ID NO:50)
(i) a nucleotide sequence which encodes the amino acid sequence:
GLRSYNYGRNLDY, (SEQ ID NO:51)
(j) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably of at least 90%, with an amino acid sequence from (a), (b), (c) or (d), and
(k) a nucleotide sequence which encodes an amino acid sequence having an equivalent ability to bind to autoantibodies against GPIIb/IIIa.
The novel nucleic acid furthermore preferably comprises a CDR1 region selected from: a nucleotide sequence which encodes the amino acid sequences NFAMS (SEQ ID NO:54), SYTMH (SEQ ID NO:55), DYALH (SEQ ID NO:56) OR SHYWS (SEQ ID NO:57) shown in Table 7a, a nucleotide sequence which encodes the amino acid sequence TYYWS (SEQ ID NO:58), a nucleotide sequence which encodes the amino acid sequences DYGMH (SEQ ID NO:59), SHTIS (SEQ ID NO:60), KYAIH (SEQ ID NO:61) or ELSMH (SEQ ID NO:62), shown in Table 7b and a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with one of the previously mentioned amino acid sequences.
Preferably, the novel nucleic acid furthermore comprises a CDR2 region selected from a nucleotide sequence which encodes the amino acid sequences GISGGGLLTHYA(D/N)SVKG (SEQ ID NO:63/SEQ ID NO:64), LISYDGSNKYYADSVKG (SEQ ID NO:111), GISWDSTSIGYADSVKG (SEQ ID NO:70) or FIYDGARTRFNPSLRS (SEQ ID NO:71) shown in Table 7a, a nucleotide sequence which encodes the amino acid sequence YIYYSGNTNYNPSLKS (SEQ ID NO:112), a nucleotide sequence which encodes the amino acid sequences AISYDGSNKYYADSVFG (SEQ ID NO:113), GITPIFGTVNYAQKFQG (SEQ ID NO:65), AISSNGGNTYYADSVKG (SEQ ID NO:72) or GFDPEDGETIYAQKFQG (SEQ ID NO:73) shown in Table 7b, and a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably of at least 90%, with one of the previously mentioned amino acid sequences.
Another part of the subject-matter of the present invention is a nucleic acid which encodes the light chain of a human antibody, or a functional derivative or a fragment thereof, and comprises a CDR3 region, selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
CSYVHSSTN, (SEQ ID NO:52)
(b) a nucleotide sequence which encodes the amino acid sequence:
QVWDNTNDQ, (SEQ ID NO:53)
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a), and
(d) a nucleotide sequence which encodes an amino acid sequence having an equivalent ability to bind to autoantibodies against GPIIb/IIIa.
Preferably, the novel nucleic acid furthermore comprises a CDR1 region selected from a nucleotide sequence which encodes the amino acid sequence TGTS SAIGNYNFVP (SEQ ID NO:66) shown in a 7a, a nucleotide sequence which encodes the amino acid sequence GGYK IGSKSVH shown in Tab. 7b (SEQ ID NO:67) and a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably of at least 90%, with the previously mentioned amino acid sequence.
In addition, the novel nucleic acid preferably furthermore comprises a CDR2 region selected from a nucleotide sequence Which encoders the amino acid sequence EGSKRPS (SEQ ID NO:68) shown in Tab. 7a, a nucleotide sequence which encodes the amino acid sequence EDSY RPS (SEQ ID NO:69) shown in Tab. 7b, and a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with the previously mentioned amino acid sequence.
Within the meaning of the present invention, the phrase xe2x80x9cfunctional derivative of a chain of a human antibodyxe2x80x9d is to be understood as meaning a polypeptide which encompasses at least a CDR3 region or the heavy and/or light chain, as defined above, and which is able, where appropriate together with the relevant complementary chain of the human antibody (or a derivative of such a chain), to form an antibody derivative which possesses a recognition specificity for an antigen which is equivalent to that possessed by the non-derivatized antibody. Preferably, such an antibody derivative has a binding constant for the relevant antigen of at least 10xe2x88x926 1/mol, preferably of at least 10xe2x88x923 1/mol.
Functional derivatives of chains of a human antibody can be prepared, for example, by using recombinant DNA techniques to delete, substitute and/or insert segments of the gene encoding the relevant polypeptide.
Single-chain antibodies, which can, for example, be composed of the variable domains of he H and L chains or one or two H chain domains and, where appropriate a constant domain, are particularly preferred functional derivatives of antibody chains or antibodies. The preparation of such constructs is described in Hoogenboom et al., Immunol. Rev. 130 (1992), 41-68; Barbas III, Methods: Companion Methods Enzymol. 2 (1991), 119 and Plxc3xcckthun, Immunochemistry (1994), Marcel Dekker Inc., Chapter 9, 210-235.
Within the meaning of the present invention, the phrase xe2x80x9cequivalent ability to bindxe2x80x9d is to be understood as being a binding affinity and/or specificity, i.e. epitope recognition, which is the same as that in the specifically disclosed sequences.
Another part of the subject-matter of the present invention is a vector which contains at least one copy of a novel nucleic acid. This vector can be a prokaryotic vector or a eukaryotic vector. Plasmids, cosmids and bacteriophages are examples of prokaryotic vectors. Such vectors are, for example, described in detail in Chapters 1 to 4 in Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor Laboratory Press. A prokaryotic vector is preferably a plasmid or a phage.
On the other hand, the vector can also be a eukaryotic vector, e.g. a yeast vector, an insect vector (baculovirus) or a mammalian vector (plasmid vector or viral vector). Examples of eukaryotic vectors are described in Sambrook at al., loc. cit., Chapter 16, and Winnacker, Gene und Klone, Eine Einfxc3xchrung fxc3xcr die Gentechnologie [Genes and clones, an introduction to genetic engineering] (1985), VCH Verlagsgesellschaft, in particular Chapters 5, 8 and 10.
Yet another part of the subject-matter of the present invention is a cell which expresses a novel nucleic acid, or a cell which is transformed with a novel nucleic acid or with a novel vector. The cell can be a prokaryotic cell (e.g. a Gram-negative bacterial cell, in particular E.coli) or a eukaryotic cell (e.g. a yeast, plant or mammalian cell). Examples of suitable cells and methods for introducing the novel nucleic acid into such cells can be found in the above literature references.
Another part of the subject-matter of the present invention is a polypeptide which is encoded by a novel nucleic acid, in particular a recombinant polypeptide. Particularly preferably, the polypeptide contains the variable domain of the H chain and/or L chain of a human antibody.
Particular preference is given to a polypeptide which exhibits antibody properties and whose subunit components are a heavy chain, or a functional derivative thereof, and a light chain, or a functional derivative thereof. The polypeptide can be composed of two separate chains or be present as a single-chain polypeptide.
Yet another part of the subject-matter of the present invention is an antibody against a novel polypeptide, which antibody is directed against a region of the polypeptide which is responsible for recognizing the antigen. This antibody can be a polyclonal antiserum, a monoclonal antibody or a fragment of a polyclonal or monoclonal antibody (e.g. a Fab, F(ab)2, Fabxe2x80x2 or F(abxe2x80x2)2 fragment). The antibody is preferably directed against the CDR3 region of the heavy and/or light antibody chain of the novel polypeptide, or a region thereof. Known methods can be used to obtain such antibodies by immunizing an experimental animal with a peptide or polypeptide which contains a novel CDR3 region and isolating the resulting polyclonal antibody from the experimental animal. In addition, monoclonal antibodies can be obtained by fusing an antibody-producing B cell from the experimental animal with a leukaemia cell in accordance with the method of Kohler and Milstein or a further development of this method. In addition, recombinant antibodies which are directed against the CDR3 region of the novel polypeptide can also be obtained by screening a suitable phagemid library, e.g. a phagemid library from a healthy human donor, with a novel polypeptide being used as the antigen.
The invention also relates to a pharmaceutical composition which comprises a nucleic acid, a vector, a polypeptide, an antibody or a cell as previously mentioned, as active component, where appropriate together with other active components and also pharmaceutically customary adjuvants, additives or excipients.
The pharmaceutical composition can be used for preparing a diagnostic or therapeutic agent. Examples of diagnostic uses are the diagnosis of AITP or of a predisposition for AITP. Another preferred diagnostic use is that of monitoring the course of the AITP disease.
The use of the pharmaceutical composition as a diagnostic agent can comprise, for example, detecting a B cell subpopulation which is expressing a novel polypeptide as the antibody. This antibody can be detected, for example, at the nucleic acid level, e.g. by means of a nucleic-acid-hybridization assay, together with prior amplification where appropriate. On the other hand, the antibody can also be detected as to the protein level by means of an immunoassay using antigens or antibodies which react specifically wish the polypeptide.
Furthermore, the novel pharmaceutical composition can also be applied in the therapeutic field, in particular for the prevention or therapy of AITP. This therapeutic use can, for example, be based on stimulating the production of anti-autoantibodies. For this, the novel autoantibody polypeptide can, for example, be administered to a patient, thereby eliciting and/or stimulating the formation of antiidiotypic antibodies. In this connection, this administration can be effected in accordance with customary immunization protocols (Fox et al., J. Pharmacol. Exp. Ther. 279 (1996), 1000-1008; Whittum-Hudson et al., Nat. Med. 2 (1996), 1116-1121; Jardieu, Curr. Opin. Imunol. 7 (1995), 779-782). On the other hand, the expression of antibody genes can be inhibited specifically by administering suitable antisense nucleic acids. The novel antiidiotypic antibody polypeptide can be administered to a patient in order to achieve direct inhibition of the autoantibody activity.
Investigations carried out on the novel autoantibody polypeptides have shown that these polypeptides are surprisingly able to inhibit the binding of fibrinogen to blood platelets. The novel autoantibody polypeptides and antiidiotypic antibody polypeptides can therefore be employed, where appropriate in combination, as agents for modulating blood coagulation, in particular for preventing a thrombosis, for example following cardiac infarctions or strokes, or in association with venous thromboses together with lung embolisms or ischaemias, etc.
Murine monoclonal antibodies, e.g. the monoclonal antibody 7E3 (cf., e.g., U.S. Pat. No. 5,440,020) or fragments thereof (e.g. the commercially available Fab fragment ReoPro(copyright)), or short synthetic peptides, have hitherto been used as fibrinogen antagonists for therapeutic purposes. However, murine monoclonal antibodies and antibody fragments suffer from the disadvantage that, as a result of their immunogenicity, they give rise to undesirable side reactions when used for treating human patients, while short peptides are generally degraded very rapidly. As compared with these known agents, the novel polypeptides have the advantage that they consist of amino acid sequences of human origin and therefore exhibit fewer undesirable side effects than do corresponding murine antibodies or antibody fragments, and that, because of their size, they are not subjected to such rapid degradation as are peptides.
The invention therefore relates to the use of a novel nucleic acid, in particular a nucleic acid which encodes an autoantibody polypeptide, of a vector which contains this nucleic acid, of a cell which is transformed with the nucleic acid or the vector, of a polypeptide which is encoded by the nucleic acid, or of a pharmaceutical composition which comprises one or more of the said substances, for preparing an agent for affecting and in particular inhibiting the binding of fibrinogen to blood platelets. Preference is given to using the agent for modulating blood coagulation, in particular for dissolving thrombi and/or for preventing the formation of thrombi. The administration of the novel pharmaceutical composition can be effected in accordance with protocols which have already been established for murine antibodies or antibody fragments.
Yet another part of the subject-matter of the invention is a process for isolating phagemid clones which express nucleic acids which encode autoantibodies against GPIIb/IIIa or encode antiidiotypic antibodies which are directed against these autoantibodies, characterized in that a phagemid library is prepared from lymphocytes from a human donor and the desired phagemid clones are isolated by affinity selection, comprising negative and positive selection steps. Preferably, the process also involves isolating antibody-encoding nucleic acids from the clones and/or using the antibody-encoding nucleic acids for expressing recombinant antibody chains or derivatives or fragments thereof.
The invention is also explained by the following examples, figures and sequence listings, in which
SEQ ID No. 1 shows the nucleotide sequence of the H chain of a novel antibody (phagemid clone PDG7), with framework region (FR)1 extending from bp 1 to 90, complement-determining region (CDR)1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 195, FR3 from bp 196 to 291, CDR3 from bp 292 to 324 and FR4 from bp 325 to 357,
SEQ ID No.2 shows the amino acid sequence corresponding to the nucleotide sequence depicted in SEQ ID No.1, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 65, FR3 from AA 66 to 97, CDR3 from AA 98 to 108 and FR4 from AA 109 to 119,
SEQ ID No.3 shows the nucleotide sequence of the L chain of a novel polypeptide (phagemid clone PDG7), with FR1 extending from bp 1 to 60, CDR1 from bp 61 to 99, FR2 from bp 100 to 144, CDR2 from bp 145 to 165, FR3 from bp 166 to 261, CDR3 from bp 262 to 294 and FR4 from bp 295 to 333,
SEQ ID No.4 shows the amino acid sequence corresponding to the nucleotide sequence given in SEQ ID No. 3, with FR1 extending from AA 1 to 20, CDR1 from AA 21 to 33, FR2 from AA 34 to 48, CDR2 from AA 49 to 55, FR3 from AA 56 to 87, CDR3 from AA 88 to 98 and FR4 from AA 99 to 11 [sic],
SEQ ID No.5 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone PDG13), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 109, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 294, CDR3 from bp 295 to 336 and FR4 from bp 337 to 369,
SEQ ID No.6 shows the amino sequence corresponding to the nucleotide sequence depicted in SEQ ID No.5, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CD2 from AA 50 to 66, FR3 from AA 67 to 98, CDR3 from AA 99 to 112 and FR4 from AA 113 to 123,
SEQ ID No.7 shows the nucleotide sequence of the L chain of a novel polypeptide (phagemid clone PGD13), with FR1 extending from bp 1 to 60, CDR1 from bp 61 to 99, FR2 from bp 100 to 144, CDR2 from bp 145 to 165, FR3 from bp 166 to 261, CDR3 from bp 262 to 294 and FR4 from bp 295 to 333,
SEQ ID No.8 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 7, with FR1 extending from AA 1 to 20, CDR1 from AA 21 to 33, FR2 from AA 34 to 48, CDR2 from AA 49 to 55, FR3 from AA 56 to 87, CDR3 from AA 88 to 98 and FR4 from AA 99 to 111,
SEQ ID No.9 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-X16), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 288, CDR3 from bp 289 to 336 and FR4 from bp 337 to 369,
SEQ ID No.10 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 9, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 96, CDR3 from AA 97 to 112 and FR4 from AA 113 to 123,
SEQ ID No. 11 shows the nucleotide sequence of the L chain of a novel polypeptide (phagemid clone AI-X16), with FR1 extending from bp 1 to 60, CDR1 from bp 61 to 102, FR2 from bp 103 to 147, CDR2 from bp 148 to 168, FR3 from bp 169 to 264, CDR3 from [lacuna] 265 to 291 and FR4 from bp 292 to 375,
SEQ ID No. 12 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 11, with FR1 extending from AA 1 to 20, CDR1 from AA 21 to 34, FR2 from AA 35 to 49, CDR2 from AA 50 to 56, FR3 from AA 57 to 88, CDR3 from AA 89 to 97 and FR4 from AA 89 to 125,
SEQ ID No. 13 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-X20), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 195, FR3 from bp 196 to 291, CDR3 from bp 292 to 333 and FR4 from bp 334 to 366,
SEQ ID No. 14 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 13, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 65, FR3 from AA 66 to 97, CDR3 from AA 98 to 111 and FR4 from AA 112 to 122,
SEQ ID No. 15 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-X39), with FR extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from pb [sic] 148 to 198, FR3 from bp 199 to 294, CDR3 from bp 295 to 339 and FR4 from 340 to 372,
SEQ ID No. 16 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 15, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 98, CDR3 from AA 99 to 113 and FR 4 from AA 114 to 124,
SEQ ID No. 17 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-X40), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 297, CDR3 from bp 298 to 339 and FR4 from bp 340 to 372,
SEQ ID No. 18 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 17, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 99, CDR3 from AA 100 to 113 and FR4 from AA 114 to 124,
SEQ ID No. 19 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-X2), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 195, FR3 from bp 196 to 291, CDR3 from bp 292 to 327 and FR4 from bp 328 to 360,
SEQ ID No. 20 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 19, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 65, FR3 from AA 66 to 97, CDR3 from AA 98 to 109 and FR4 from AA 110 to 120,
SEQ ID No. 21 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-B14), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 294, CDR3 from bp 295 to 336 and FR4 from bp 337 to 369;
The following variations in the sequence were also found: a C can be present at position 7, while a G can be present at position 9, a G at position 13, a G at position 15, an A at position 91, a G at position 92, a C at position 98, a T at position 149, an A at position 205, an A at position 228, an A at position 251, a T at position 253 and/or an A at position 284. The consequence of this is that, in the amino acid sequence (cf. SEQ ID No. 22), a Q can be present at position 3, while a V can be present at position 5, an S at position 31, an A at position 33, a V at position 50, a T at position 69, a K at position 76, an N at position 84, an S at position 85 and/or a Y at position 95.
SEQ ID No. 22 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 21, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 98, CDR3 from AA 99 to 112 and FR4 from AA 113 to 123,
SEQ ID No. 23 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-B18), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 294, CDR3 from bp 295 to 333 and FR4 from bp 334 to 366;
The following variations in the nucleotide sequence were also found: thus, a C can be present at position 7, while a G can be present at position 13, a C at position 16, an A at position 56, a T at position 94, a G at position 97, a T at position 155, a C at position 173, a T at position 223, a T or a C at position 252, a G at position 261, a G at position 267, an A at position 271, a C at position 275 and/or a G at position 277. The consequence of this is that, in the corresponding amino acid sequence (cf. SEQ ID No. 24), a Q can be present at position 3, while a V can be present at position 5, a Q at position 6, a K at position 19, a Y at position 32, an A at position 33, an I at position 52, an A at position 58, an S at position 75, an S at position 84, an R at position 87, an E at position 89, a T at position 91, an A at position 92 and/or a V at position 93.
SEQ ID No. 24 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 23, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 98, CDR2 from AA 99 to 111 and FR4 from AA 112 to 122,
SEQ ID No. 25 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-B24), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 294, CDR3 from bp 295 to 330 and FR4 from bp 331 to 363;
The following variations in the nucleotide sequence were also found: a C can be present at position 7, while a G can be present at position 9, a G at position 13, a G at position 15, a G at position 31, an A at position 46, a G at position 67, a G at position 89, a G at position 92, a C at position 93, a G at position 98, a G at position 102, a G at position 140, a C at position 141, a G at position 145, a T at position 149, a T at position 157, an A at position 158, a G at position 160, an A at position 166, an A at position 173, a T at position 235, an A at position 251, a C at position 290 and/or an A at position 293. The consequence of this is that, in the corresponding amino acid sequence (cf. SEQ ID No. 26), a Q can be present at position 3, while a V can be present at position 5, a V at position 11, an R at position 16, an A at position 23, an S at position 30, an S at position 31, a G at position 33, an M at position 34, a W at position 47, an A at position 49, a V at position 50, a Y at position 53, a D at position 54, an S at position 56, a K at position 58, an L at position 79, an N at position 84, an A at position 97 and/or a K at position 98.
SEQ ID No. 26 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 25, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 98, CDR3 from AA 99 to 110 and FR4 from AA 111 to 121,
SEQ ID No. 27 shows the nucleotide sequence of the L chain of a novel polypeptide (phagemid clone AI-B24), with FR1 extending from bp 1 to 60, CDR1 from bp 61 to 96, FR2 from bp 97 to 138, CDR2 from bp 139 to 159, FR3 from bp 160 to 255, CDR3 from bp 256 to 282 and FR4 from bp 283 to 366;
The following variations in the nucleotide sequence were also found: a C or a T can be present at position 4, while a G can be present at position 37, an A at position 40, a G at position 50, an A at position 67, a T at position 72, an A at position 133, a T at position 136, a T or a C at position 138, a G at position 148, a T at position 160, a T at position 161, a T or a C at position 162, a C at position 200, a T at position 217, a G at position 218, an A or a C at position 220, a G at position 269, a T at position 271, a G at position 272, a G at position 275 and/or a T or a C at position 282. The consequence of this is that, in the a corresponding amino acid sequence (cf. SEQ ID No. 28), an L can be present at position 2, while a G can be present at position 13, a K at position 14, an R at position 17, an N at position 23, an N at position 24, an I at position 45, a Y at position 47, a D at position 50, an F at position 54, a T at position 67, an S at position 73, an R at position 74, an S at position 90, an S at position 91, an S at position 92 and/or an H at position 94.
SEQ ID No. 28 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 27, with FR1 extending from AA 1 to 20, CDR1 from AA 21 to 32, FR2 from AA 33 to 46, CDR2 from AA 47 to 53, FR3 from AA 54 to 85, CDR3 from AA 86 to 94 and FR4 from An 95 to 122,
SEQ ID No. 29 shows the nucleotide sequence of the H chain of a novel polypeptide (phagemid clone AI-B38), with FR1 extending from bp 1 to 90, CDR1 from bp 91 to 105, FR2 from bp 106 to 147, CDR2 from bp 148 to 198, FR3 from bp 199 to 294, CDR3 from bp 295 to 333 and FR4 from bp 334 to 366;
The following variations in the nucleotide sequence were also found: a C can be present at position 7, while a G can be present at position 9, a G at position 13, an A at position 15 and/or a C at position 16. The consequence of this is that, in the corresponding amino acid sequence, a Q can be present at position 3, while a V can be present at position 5 and/or a Q can be present at position 6, and
SEQ ID No. 30 shows the amino acid sequence of the nucleotide sequence depicted in SEQ ID No. 29, with FR1 extending from AA 1 to 30, CDR1 from AA 31 to 35, FR2 from AA 36 to 49, CDR2 from AA 50 to 66, FR3 from AA 67 to 98, CDR3 from AA 99 to 111 and FR4 from AA 112 to 122.